Connective-tissue-based or dermal-tissue-based grafts/implants

ABSTRACT

The present invention is directed to a composition comprising a matrix suitable for implantation in humans, comprising defatted, shredded, allogeneic human muscle tissue that has been combined with an aqueous carrier and dried in a predetermined shape. Also disclosed is a tissue graft or implant comprising a matrix suitable for implantation in humans, comprising defatted, shredded, allogeneic human muscle tissue that has been combined with an aqueous carrier and dried in a predetermined shape. The composition and/or tissue graft or implant of the invention is usable in combination with seeded cells, a tissue growth factor, and/or a chemotactic gent to attract a desired cell.

This application is a continuation of U.S. patent application Ser. No.15/192,368, filed Jun. 24, 2016, now pending, which is a continuation ofU.S. patent application Ser. No. 14/284,918, filed May 22, 2014, nowU.S. Pat. No. 9,398,948, which is a continuation of U.S. patentapplication Ser. No. 13/815,184, filed Feb. 7, 2013, now U.S. Pat. No.8,747,467, which is a continuation of U.S. patent application Ser. No.12/807,599, filed Sep. 9, 2010, now U.S. Pat. No. 8,394,141, which is acontinuation of U.S. patent application Ser. No. 11/949,687, filed Dec.3, 2007, now U.S. Pat. No. 7,883,541, which is a continuation of U.S.patent application Ser. No. 11/480,711, filed Jul. 3, 2006, nowabandoned, which is a continuation of U.S. patent application Ser. No.10/793,976, filed Mar. 5, 2004, now U.S. Pat. No. 7,131,994, which is acontinuation-in-part of U.S. patent application Ser. No. 10/754,310,filed Jan. 9, 2004, now U.S. Pat. No. 7,001,430.

BACKGROUND OF THE INVENTION

The present invention is directed to the field of biocompatible matricesfor use in forming devices for implantation in animals and humans. Moreparticularly, the present invention is directed to an implantablecomposition or a tissue graft/implant formed from an allogeneicbiocompatible human muscle matrix that is capable of carrying otherimplantable materials or that can be formed into a plurality of tissueimplants or compositions having different properties and differentshapes. The present invention is useful because it provides animplantable composition or device that is versatile in its ability to beformulated into a variety of implants or grafts that are useful in thetreatment of a variety of medical conditions in patients.

In the field of biomedical implants, devices have been made that rangefar afield from the biological components found in the human body. Forexample, many devices that are intended as bone substitutes are madefrom metals such as titanium, or biocompatible ceramics. A problem insuch instances is that they have different material properties than thehost tissue causing the devices to loosen at the interface between thehost tissue and the device itself

One solution to the problem was the use of allograft bone in place ofmetal or ceramic implants. Under the proper conditions and under theinfluence of osteogenic substances, implants made of allograft bone canact as the scaffolding for remodeling by the host. Such implantsfunction by being both structurally and biologically similar to the hosttissue. Further, they allow cellular recruitment through the naturalopenings in the matrix and allow the graft to be replaced by naturalhost bone. While allograft bone is very useful, it is limited by theintended clinical use. Thus, it is particularly useful for spinalfusions where the spacings between the vertebrae are relatively fixedand well known. However, injuries come in a variety of shapes and sizesthat present a logical limitation on the availability of an ideal graftto fill the defect. Moreover, availability, donor demographics and costfurther limit the usefulness of allograft bone. Accordingly, there is aneed in the art—for an implantable biocompatible matrix that can beformulated into a variety of shapes and sizes and that can act asscaffolding to allow the infiltration of native regenerative cells thatwill lay down a natural replacement structure in the shape of theimplant.

Another example area where biocompatible implants are important is inreplacement skin for burn victims. Histocompatibility, remodeling andsafety are considerable problems in utilizing allograft skin. To avoidthis problem and the shortage of viable donor skin, a surgeon oftenremoves skin from another part of the patient and transplants it to thearea of need. While such skin is non-antigenic, it causes significantmorbidity to the patient at the site of removal. Moreover, dependingupon the size of the wound or burn, there may not be sufficient skin onthe patient to satisfy the need. To alleviate this problem, at least onecompany will culture the patient's skin cells on a collagen matrix toform a transplantable layer of skin. However, the culture time isrelatively extensive and the patient's wound or burn is exposed whileawaiting the graft. Moreover, the grafts generated in this way do notmimic normal skin, which is composed of multiple cell types andstructures. Accordingly, there is a need in the art for an implantablebiocompatible matrix that can be formulated into a sheet and cut to sizeand that can act as scaffolding to allow the infiltration of a varietyskin cells from adjacent tissue that will lay down a compatible andnatural replacement structure in the shape of the implant, whileabsorbing the implant itself.

It is an object of the present invention to prepare a matrix frombiological tissue that has the ability to be formulated into a varietyof forms and shapes that can participate in the correction of a varietyof pathologies such as those described above.

SUMMARY OF THE INVENTION

The applicants have discovered a composition that provides abiocompatible, non-antigenic matrix and scaffolding material for tissueregeneration in humans. In its simplest form, the present invention isdirected to a composition comprising a matrix suitable for implantationin humans, comprising defatted, shredded, allogeneic human muscle tissuethat has been combined with an aqueous carrier and dried. Typically, thecomposition of the present invention is sufficiently dried to so as tobe able to be handled. More typically, it is dried to a moisture contentof about 3% or less.

In another aspect, the present invention is directed to a tissuegraft/implant suitable for implantation in humans comprising a matrix ofdefatted, shredded, allogeneic human muscle tissue that has beencombined with an aqueous carrier and dried in a predetermined shape.Typically, the shape of the tissue graft/implant of the presentinvention includes a strip, a sheet, a disc, a molded 3D shaped object,a plug, a sponge, and a gasket. Typically, the composition of thepresent invention is sufficiently dried to so as to be able to behandled. More typically, it is dried to a moisture content of about 3%or less.

Any human muscle is suitable for use in the compositions or tissuegraft/implant of the present invention, including smooth muscle andstriated muscle. Preferably, the human muscle tissue that is employed isstriated muscle, such as skeletal muscle or cardiac muscle. Morepreferably, the muscle tissue employed is skeletal muscle tissue.

Any of the compositions of tissue graft/implants of the presentinvention may include collagen fibers, growth factors, antibiotics,cells, or particles such as demineralized bone matrix (DBM), mineralizedbone matrix, cortical cancellous chips (CCC), crushed cancellous chips,tricalcium phosphate, hydroxyapatite, or biphasic calcium phosphate(wherein the latter is the combination of tricalcium phosphate andhydroxyapatite) or a combination thereof

A composition or tissue graft/implant of the present invention that isparticularly suited for treating bone trauma, bone disease or bonedefects, for providing artificial arthrodeses, or for other treatmentwhere new bone formation is desired, further comprises particles of DBM,mineralized bone matrix, CCC, crushed cancellous chips, tricalciumphosphate, hydroxyapatite, or biphasic calcium phosphate dispersed inthe matrix.

A preferred composition or tissue graft/implant of the present inventionthat is particularly suited for treating bone trauma, bone disease orbone defects, for providing artificial arthrodeses, or for othertreatment where new bone formation is desired, further comprisesparticles of DBM, mineralized bone matrix, CCC, crushed cancellouschips, tricalcium phosphate, hydroxyapatite, or biphasic calciumphosphate dispersed in the matrix, in combination with a therapeuticallyeffective amount of a growth factor selected from the group consistingof bone morphogenic protein (BMP), LIM mineralization protein (LMP) andRUNX-2.

A preferred growth factor is BMP. BMP is a well-known naturallyoccurring bone protein and may be obtained by extraction from freshbone. Methods for isolating BMP from bone are described in U.S. Pat. No.4,294,753 to Urist and Urist et al., PNAS 371, 1984. Often BMP isobtained by packing fresh fragments of bone into a cavity in an implantthat is designed for receiving such packing. However, the amount of BMPin such packing is variable. Therefore, it is preferred that the BMP bea recombinant human BMP such that its activity is known. Recombinanthuman BMPs are commercially available or prepared as described and knownin the art, e.g., in U.S. Pat. No. 5,187,076 to Wozney et al.; U.S. Pat.No. 5,366,875 to Wozney et al.; U.S. Pat. No. 4,877,864 to Wang et al.;U.S. Pat. No. 5,108,932 to Wang et al.; U.S. Pat. No. 5,116,738 to Wanget al.; U.S. Pat. No. 5,013,649 to Wang et al.; U.S. Pat. No. 5,106,748to Wozney et al; and PCT Patent Nos. WO93/00432 to Wozney et al.;W094/2693 to Celeste et al.; and WO94/26892 to Celeste et al., all ofwhich are hereby incorporated herein by reference in their entirety.Recombinant human BMP-2 (rhBMP-2), recombinant human BMP-4 (rhBMP-4),recombinant human BMP-7 (rhBMP-7) or heterodimers thereof are morepreferred. rhBMP-2 is most preferred.

The amino acid sequence of the RUNX-2 protein and vectors suitable forexpressing the protein are disclosed in co-pending U.S. patentapplication Ser. No. 10/437,171, filed May 13, 2003, and incorporatedherein in its entirety.

Other suitable tissue growth factors for use in combination with thecomposition and with the tissue graft/implant of the present inventioninclude transforming growth factor-β (TGF-β), a fibroblast growth factor(FGF) such as FGF-1 to FGF-12, platelet-derived growth factor (PDGF),and insulin-like growth factor (ILGF). All of these factors are wellknown in the art.

The composition and the tissue graft/implant of the present inventioncomprise a matrix that is formed from the fibers of defatted, shredded,allogeneic human muscle tissue. This fibrous structure is advantageousbecause the resulting matrix that is formed is porous and particularlywell suited both for the infiltration by colonizing cells (e.g.,osteoconduction), and for the storage and slow release of seeded cells,growth factors (as described above), and chemotactic agents to attractdesired cells (e.g., osteoinduction).

Thus, it is also within the scope of the present invention that thecomposition or implant/tissue graft of the present invention be combinedwith seeded cells, a tissue growth factor, or a chemotactic agent, or acombination thereof.

When the composition or tissue graft/implant of the present invention isto be used as a tissue graft for skin, it is optionally seeded withdermatocytes, more typically with dermatocytes and melanocytes.

When the composition or tissue graft/implant of the present invention isto be used to treat bone trauma, disease and defects, for artificialarthrodeses and for other treatment where new bone formation is desired,it is optionally seeded with osteogenic cells. Preferably, thecomposition or tissue graft/implant of the present invention is seededwith stem cells that will provide a natural distribution of the nativecells necessary for restoration of the injury or defect at the site ofimplantation.

The tissue grafts and implants of the present invention exhibit a greatdegree of tensile strength. They are readily stitchable and retain amajority of their tensile strength even when rehydrated. In addition,upon hydration, tissue grafts and implants of the present invention aremoldable and suitable for filling in irregular gaps or holes in thetissue to be repaired. Typically, the hydrated tissue is press fitted bythe surgeon into the defect or cavity to be filled.

It is also within the scope of the present invention that the tissuegraft/implant of the present invention be utilized with a load-bearingmember used in a spinal fusion. Suitable load bearing members includehollow spinal cages, hollow dowels, C-shaped and D-shaped spacers andother devices known in the art, having a pocket, chamber or othermechanism for retaining the tissue graft/implant of the presentinvention. Typically, the load-bearing member has a compressive strengthof at least about 1,000 N. More typically, when utilized between lumbarvertebrae, the load-bearing member has a compressive strength of 3,000to 11,000 N. When utilized between cervical vertebrae, the load bearingmember has a compressive strength of about 1,000 to 3,000 N. Suitableload bearing members are known in the art and described in multiple U.S.patents, including, for example in U.S. Pat. Nos. 5,522,899, 5,785,710,5,776,199 and 5,814,084, 6,033,438, 6,096,081, each of which is herebyincorporated by reference in its entirety

It was unexpectedly discovered that the skeletal muscle-basedimplantable devices and compositions of the present invention havehemostatic action and cause platelet activation. This is a particularadvantage during surgical procedures wherein patient bleeding is awell-known problem.

In its second aspect, the present invention is directed to a method formaking a (muscle-based) composition or a tissue implant suitable fortreating an injury or a surgical or medical condition in a humanpatient, wherein the tissue implant comprises a matrix of allogeneichuman muscle. In this embodiment, the method comprises the steps of:

-   -   i. removing the fat and soluble proteins from allogeneic or        xenogeneic mammalian muscle tissue;    -   ii. lyophilizing the muscle tissue from step (i);    -   iii. shredding the lyophilized muscle tissue;    -   iv. mixing the shredded muscle tissue in an aqueous carrier to        form a muscle tissue slurry having a viscosity within the range        of 1 centistoke to 20,000 centistokes;    -   v. transferring the muscle tissue slurry to an appropriate        shaped mold; and    -   vi. drying the slurry in the mold to form the correspondingly        shaped tissue implant.

In the above method, the matrix of allogeneic human muscle comprisesfrom about 1% to about 100% of the final weight of the composition orimplant, more typically, from 50% to about 99% of the final weight ofthe implant, even more typically from 75% to about 99% of the finalweight of the implant.

In another embodiment, the method of making a composition or tissueimplant of the present invention further comprises combining theallogeneic human muscle with a demineralized bone matrix (DBM), corticalcancellous chips (CCC), tricalcium phosphate, hydroxyapatite, orbiphasic calcium phosphate and/or shredded allogeneic human tendon. Theaddition of any of these components increases the viscosity of theintermediate slurry. When the tissue implant of the present inventioncontains DBM, mineralized bone matrix, CCC, crushed cancellous chips,tricalcium phosphate, hydroxyapatite, or biphasic calcium phosphate or acombination thereof, the resulting tissue implant is osteogenic andparticularly suited for repairing bone. The implant of this embodimentalso exhibits improved dimensional stability and ability to hold shapeduring drying, rehydration and handling. In one variation of the aboveembodiment, the DBM, mineralized bone matrix, CCC, crushed cancellouschips, tricalcium phosphate, hydroxyapatite, or biphasic calciumphosphate or a combination thereof are dispersed equally or randomlythroughout the matrix. In another variation, the DBM, mineralized bonematrix, CCC, crushed cancellous chips, or a combination thereof aresandwiched between layers of the matrix to form a laminate implant. Ineither of the above referenced embodiments, the DBM, mineralized bonematrix, CCC, crushed cancellous chips, tricalcium phosphate,hydroxyapatite, or biphasic calcium phosphate or a combination thereoftypically constitute from 1% to 99%, more typically 50% to 99%, mosttypically from 90% to 99% of the dry weight of the composition orimplant.

When the tissue implant contains tendon, it is tougher, and strongerthan the digested human muscle matrix alone and is particularly suitedas a dressing for a wound or burn that will become infiltrated with skincells and allow for development of a replacement skin layer that willcover the wound or burn. The ratio of tendon to intermediate compositionranges from 1:99 to 99:1 by dry weight. Typically, the range is 10:90 to90:10; more typically, the range is 25:75 to 75:25. While the abovediscussion is in relation to “tendon,” which is a preferred source ofcollagen for this invention, it is intended that any collagen source beused, including fascia, ligament, or dermis.

After the mixing step, the resulting intermediate composition (thedigested allogeneic human muscle slurry) of the present invention isoptionally degassed, by pouring the slurry into plates or tubes, andcentrifuging them to eliminate any entrapped air and produce a higherdensity slurry.

Alternatively, the slurry is poured into a mold for formation of animplantable tissue matrix of any size or shape. As noted above, theslurry can be combined with other agents, such as DBM, CCC or a collagen(e.g., tendon, fascia) slurry before being poured into the mold. Toproduce an implantable film, a thin layer of the slurry is poured in aflat plate and the slurry is either air dried, air dried with positiveairflow, or dried in an oven, preferably a convection oven. To produce asponge, a gasket or an implantable shape, the slurry (neat or amended)is poured into a mold of the appropriate shape, frozen (to retain itssize), and lyophilized. The resulting dried implantable film or shape isthen ready for packaging and final sterilization.

Prior to implantation, the freeze dried composition, tissue graft orimplant of the present invention is removed from its sterile packagingand rehydrated by contacting with water, saline, blood, plasma, abuffered solution, or any other suitable liquid. Preferably, therehydrating liquid contains a growth factor or a chemotactic agent asdiscussed above.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The patent or patent application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fees.

FIG. 1 is a photograph showing the fluffy fibrous texture of shredded,defatted allogeneic human muscle for use in making the intermediatecomposition (muscle slurry) of the present invention.

FIGS. 2A-2C are photographs of tissue implants in the form of a spongethat were made from the muscle slurry of the present invention.

FIGS. 3A-3B are photographs of a three-dimensional molded tissue implantmade from the muscle slurry of the present invention. FIG. 3A. is a sideview. In FIG. 3B, the implant is rotated 90° to show the hole that wasmolded in the center.

FIGS. 4A and 4B are photographs of tissue implants/grafts in the form ofa thin film that were made from the muscle slurry of the presentinvention. In FIG. 4A, the film was formed from the muscle slurrywithout an additive. In FIG. 4B, the film was made from a mixed slurrycomprising a 50:50 ratio of muscle tissue to tendon tissue.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has multiple embodiments. In its first embodiment,the present invention is directed to a composition that provides abiocompatible, non-antigenic matrix and scaffolding material for tissueregeneration in humans. More specifically, the present invention isdirected to a composition comprising a matrix suitable for implantationin humans, comprising defatted, shredded, allogeneic human muscle tissuethat has been combined with an aqueous carrier and dried. Typically, thecomposition of the present invention is sufficiently dried to so as tobe able to be handled. More typically, it is dried to a moisture contentof about 3% or less.

To be suitable for implantation in humans, the composition (andimplants) of the present invention must be treated to remove anyantigenic proteins, which may generate a rejection of the implant. Italso must be treated to remove any bacteria and viruses. Suitableprocesses for removing antigenic proteins and sterilizing to neutralizeany bacteria and viruses are known in the art. See U.S. Pat. No.5,846,484, entitled “Pressure flow system and method for treating afluid permeable workpiece such as a bone,” which issued to Scarborough,et al. on Dec. 8, 1998. In the present case, the applicants utilized theassignees' well known method for defatting tissue, which also has theadded benefit of removing blood, cellular debris, and soluble andantigenic proteins, by subjecting the muscle tissue to alternatingcycles of pressure and vacuum in the sequential presence of solvents,such as isopropyl alcohol, hydrogen peroxide and a detergent. Theseassignee's processes also neutralize any bacteria and viruses. Theseprocesses are disclosed in full detail in assignee's U.S. Pat. No.6,613,278, entitled “Tissue Pooling Process,” which issued to Mills etal., on Sep. 2, 2003; U.S. Pat. No. 6,482,584, entitled “Cyclic implantperfusion cleaning and passivation process,” which issued to Mills, etal. on Nov. 19, 2002; and U.S. Pat. No. 6,652,818, entitled “ImplantSterilization Apparatus,” which issued to Mills et al., on Nov. 25,2003, all of which are incorporated herein by reference in theirentirety.

In its second aspect, the present invention is directed to a tissuegraft/implant suitable for implantation in humans comprising a matrix ofdefatted, shredded, allogeneic human muscle tissue that has beencombined with an aqueous carrier and dried in a predetermined shape. Thetissue graft/implant of the present invention is made in various shapesincluding that of a strip, a sheet, a disc, a molded 3D shaped object, aplug, a sponge, and a gasket. A preferred shape is a sponge. Any ofthese objects may include a cavity, a pouch, a hole, a post, a hook, ora suture. Any of these objects may be made with or without the additionof demineralized bone matrix (DBM), mineralized bone matrix, corticalcancellous chips (CCC), crushed cancellous chips, lyophilized andshredded muscle or collagen fibers, growth factors, antibiotics, stemcells, or other additives.

Preferably, the human muscle tissue that is employed in the compositionand tissue graft/implant of the present invention is striated muscle,such as skeletal muscle or cardiac muscle, more preferably, skeletalmuscle tissue.

A composition or tissue graft/implant of the present invention that isparticularly suited for treating bone trauma, bone disease or bonedefects, for providing artificial arthrodeses, or for other treatmentwhere new bone formation is desired, further comprises particles of DBM,mineralized bone matrix, CCC, crushed cancellous chips, tricalciumphosphate, hydroxyapatite, or biphasic calcium phosphate dispersed inthe matrix.

Typically, particles of DBM or CCC or both are added to the compositionor to the tissue graft/implant of the present invention when thecomposition or implant is being used as a scaffold to obtain boneregeneration or remodeling at the site of implantation. In thisembodiment, the mean size of the particles of DBM and mineralized bonematrix are typically from 150 microns to 900 microns, more typicallyfrom 250 microns to 800 microns, most typically from 400 to 500 microns.The CCC and crushed cancellous chips are utilized in a larger particlesize than the DBM. The mean particle size for CCC is typically 0.5 mm to5 mm, more typically 1 mm to 3 mm, and even more typically from 1.5 mmto 2.5 mm.

A preferred composition or tissue graft/implant of the present inventionthat is particularly suited for treating bone trauma, bone disease orbone defects, for providing artificial arthrodeses, or for othertreatment where new bone formation is desired, further comprisesparticles of DBM, mineralized bone matrix, CCC, crushed cancellouschips, tricalcium phosphate, hydroxyapatite, or biphasic calciumphosphate dispersed in the matrix, in combination with a therapeuticallyeffective amount of a growth factor selected from the group consistingof bone morphogenic protein (BMP), LIM mineralization protein (LMP) andRUNX-2.

A preferred growth factor is BMP. The term BMP encompasses a family ofwell-known naturally occurring proteins (BMP-2, BMP-3, BMP-4, BMP-5,BMP-6, BMP-7, BMP-13 and BMP-14) that are involved in skeletalformation. BMP2 or BMP2A is a 396 amino acid (aa) dimeric protein inhuman (chr 20p12) that belongs to the TGF-beta superfamily ofstructurally related signaling proteins. It is involved in cartilage andbone formation during embryogenesis, but may have additional functionsin morphogenesis as implied by its expression in various organs andembryonic tissues. It is abundant in bone, lung, spleen, and colon.Recombinant human BMP-2 (rhBMP-2) consists of two 115 amino acid chainsthat lack the natural N-terminus and that are bonded by a disulfide bondinto a homodimeric protein with an apparent molecular weight of 26 kDa.The rh-BMP-2, lacking the natural N-terminus, has 15-20 times specificactivity of the wild-type BMP-2. Methods for isolating BMP from bone aredescribed in U.S. Pat. No. 4,294,753 to Urist and Urist et al., PNAS371, 1984. Often BMP is obtained by packing fresh fragments of bone intoa cavity in an implant that is designed for receiving such packing.However, the amount of BMP in such packing is variable. Therefore, it ispreferred that the BMP be a recombinant human BMP such that its activityis known. Recombinant human BMPs are commercially available as describedbelow or prepared as described and known in the art, e.g., in U.S. Pat.No. 5,187,076 to Wozney et al.; U.S. Pat. No. 5,366,875 to Wozney etal.; U.S. Pat. No. 4,877,864 to Wang et al.; U.S. Pat. No. 5,108,932 toWang et al.; U.S. Pat. No. 5,116,738 to Wang et al.; U.S. Pat. No.5,013,649 to Wang et al.; U.S. Pat. No. 5,106,748 to Wozney et al; andPCT Patent Nos. WO93/00432 to Wozney et al.; W094/2693 to Celeste etal.; WO94/26892 to Celeste et al., and Hogan et al., (1996) Gene Dev.,10:1580-1594; all of which are hereby incorporated herein by referencein their entirety.

BMP3/Osteogenin/BMP-3A is a 472aa protein in human (chr 4p14-q21),highly expressed in lung, ovary and small intestine. Its function isinvolved in the cartilage and bone formation. BMP3 and BMP2 genes map toconserved regions between human and mouse. Sequences or methods ofisolating BMP-3 are disclosed in Kawabata, M et al. (1998) Cytokine andGrowth Factor Reviews 9: 49-61; Ebendal, T et al. (1998), J. Neurosci.Res. 51: 139-146; Reddi, A. H (1998), Nature Biotechnology 16: 247-252;Daluiski, A et al. (2001), Nature Genetics 27: 84; and Bahamonde, M. Eet al. (2001) Bone and Joint Surgery 83-A (suppl 1): S156, each of whichis incorporated herein by reference.

BMP-3B/GDF-10, a 478aa protein in human (Chr 10q11.22), belongs to agroup of proteins that can induce endochondral bone formation in adultanimals, it is closely related in sequence to BMP3 with 44% homology.The amino acid sequences of human and rat BMP-3b precursor proteins are83% similar, whereas the mature proteins show 98% identity. BMP-3B ismainly expressed in femur, brain, lung, pancreas and testis. Sequencesor methods of isolating BMP-3 are disclosed in Hino. J et al. (1996)Biochem Biophysic. Res. Commun. 223 (2), 304-310; Cunningham, N. S. etal. (1995) Growth Factors 12 (2), 99-109; A. H (1998), NatureBiotechnology 16: 247-252; and Daluiski, A. et al. (2001), NatureGenetics 27: 8, each of which is incorporated herein by reference.

BMP4/BMP-2B is a 408aa protein (Chr.14q22) and vital regulatory moleculethat functions throughout development in mesoderm induction, toothdevelopment, limb formation, bone induction, and fracture repair. Inhuman it is expressed in lungs, kidney and is secreted into theextracellular matrix. Sequences or methods of isolating BMP-4 aredisclosed in Hogan et al., (1996) Gene Dev., 10:1580-1594; Kawabata, Met al. (1998) Cytokine and Growth Factor Reviews 9: 49-61; Ebendal, T etal. (1998), J. Neurosci. Res. 51: 139-146; Reddi, A. H (1998), NatureBiotechnology 16: 247-252; Daluiski, A et al. (2001), Nature Genetics27: 84; Bahamonde, M. E et al. (2001) Bone and Joint Surgery 83-A (suppl1): S156, each of which is incorporated herein by reference.

BMP5 is a 454aa protein mainly expressed in lungs and liver. (Chr 6).The Bmp5 gene is expressed at the earliest stages of skeletaldevelopment in small, local patterns that prefigure the shapes of futureskeletal elements. Based upon a high degree of amino acid sequencehomology, BMP5, BMP6, and BMP7 constitute a subfamily within the BMPs.Sequences or methods of isolating BMP-5 are disclosed in Kawabata, M etal. (1998) Cytokine and Growth Factor Reviews 9: 49-61, Ebendal, T etal. (1998), J. Neurosci. Res. 51: 139-146; Reddi, A. H (1998), NatureBiotechnology 16: 247-252, each of which is incorporated herein byreference.

BMP6 or VGR1 is a 57 kD protein with 513aa in human (chr 6p24).Increased production of BMP6 is mediated by the skeletal effects ofestrogen on bone and cartilage. BMP-6 differs from other members of theBMP family by its concentration in cartilage of the fetus. Sequences ormethods of isolating BMP-6 are disclosed in Kawabata, M et al. (1998)Cytokine and Growth Factor Reviews 9: 49-61; Ebendal, T et al. (1998),J. Neurosci. Res. 51: 139-146; Reddi, A. H (1998), Nature Biotechnology16: 247-252; and Celeste, A et al. (1990) PNAS. 87: 9843-9847, each ofwhich is incorporated herein by reference.

BMP7 or OPla 431-amino acid polypeptide (chr 20) that includes asecretory signal sequence, expressed in kidney, bladder and brain. BMP-7induces cartilage and bone formation. It also plays a role in calciumregulation and bone homeostasis. Sequences or methods of isolating BMP-7are disclosed in Sampath, T. K et al. (1990) JBC 265: 13198-13205,Reddi, A. H et al. (1998), Nature Biotechnology 16: 247-252; Helder, M.N et al (1995) J. Histochem. Cytochem 43: 1035-1044; and Godin, R. E etal. (1998) Development 125: 3473-3482, each of which is incorporatedherein by reference.

BMP13/CDMP-2/GDF-6 is a cartilage derived morphogenetic protein (CDMP)with 436aa precursor sequence, that is cleaved to a 120aa polypeptidemature chain Like all BMPs, it exists as homodimer subunits linked witha disulphide bond. This protein is predominantly expressed in long bonesduring human embryonic development. Sequences or methods of isolatingBMP-13 are disclosed in Chang, S. C et al. (1994) JBC Vol. 269 (45),28227-28234; Paralkar V. M et al. (1998) JBC 273 (22) 13760-13767;Tomaski S M et al. (1999) Arch Otolaryngol Head Neck Surg. 125 (8)901-906.

BMP14/CDMP-1/GDF-5 is a 501aa precursor protein (chr 20q11.2) with a121aa mature chain. It is predominantly expressed in long bones duringembryonic development and is involved in bone formation. Sequences ormethods of isolating BMP-14 are disclosed in Chang, S. C et al. (1994)JBC Vol. 269 (45), 28227-28234; Paralkar V. M et al (1998) JBC 273 (22)13760-13767; and Tomaski S M et al. (1999) Arch Otolaryngol Head NeckSurg. 125 (8) 901-906, each of which is incorporated herein byreference.

Each of the above described BMP's is commercially available from avariety of sources. Human recombinant BMP-2 is commercially availablefrom a plurality of sources, including Genetics Institute, Inc.,Andover, Mass., Abbott Laboratories, North Chicago Ill., and YamanouchiPharmaceutical Co., Japan. Recombinant BMP-3 to rBMP14 are commerciallyavailable from Alpha Diagnostics, San Antonio Tex.

The preferred BMPs are recombinant human BMP-2 (rhBMP-2), recombinanthuman BMP-4 (rhBMP-4), recombinant human BMP-7 (rhBMP-7) andheterodimers thereof. rhBMP-2 is most preferred.

The amino acid sequence of the RUNX-2 protein and vectors suitable forexpressing the protein are disclosed in co-pending patent applicationU.S. Ser. No. 10/437,171, filed May 13, 2003, and incorporated herein inits entirety.

Other suitable tissue growth factors for use in combination with thecomposition and with the tissue graft/implant of the present inventioninclude transforming growth factor-β (TGF-β), a fibroblast growth factor(FGF) such as FGF-1 to FGF-12, platelet-derived growth factor (PDGF),and insulin-like growth factor (ILGF). All of these factors are wellknown in the art.

The composition and the tissue graft/implant of the present inventioncomprise a matrix that is formed from the fibers of defatted, shredded,allogeneic human muscle tissue. This fibrous structure is advantageousbecause the resulting matrix that is formed is porous and particularlywell suited both for the infiltration by colonizing cells, and for thestorage and slow release of seeded cells, tissue growth factors (asdescribed above), and chemotactic agents to attract desired cells.

Thus, it is also within the scope of the present invention that thecomposition or implant/tissue graft of the present invention be combinedwith seeded cells, a tissue growth factor, or a chemotactic agent, or acombination thereof.

When the composition or tissue graft/implant of the present invention isto be used as a tissue graft for skin, it is optionally seeded withfibroblasts, more typically with fibroblasts, and melanocytes. In thisembodiment, it is preferred that the composition or tissue graft furthercomprise a growth factor suitable for inducing the growth of thefibroblasts and/or melanocytes. Suitable growth factors include afibroblast growth factor (FGF), such as FGF-1 of FGF-2, epidermal growthfactor, transforming growth factor-a (TGF-a), transforming growthfactor-β (TGF-β), platelet derived growth factor (PDGF) or a combinationthereof. Each of these growth factors are well known in the art andcommercially available.

When the composition or tissue graft/implant of the present invention isto be used to treat bone trauma, disease and defects, for artificialarthrodeses and for other treatment where new bone formation is desired,it is optionally seeded with osteogenic cells. Preferably, thecomposition or tissue graft/implant of the present invention is seededwith stem cells that will provide a natural distribution of the nativecells necessary for restoration of the injury or defect at the site ofimplantation.

The implant/graft of the present invention comprising a matrix ofdigested allogeneic human muscle was made in various shapes includingthat of a strip, a sheet, a film, a disk, a molded 3D shaped object, aplug, a sponge, and a gasket. Any of these objects may include a cavity,a pouch, a hole, a post, a hook, or a suture. Any of these objects maybe made with or without the addition of DBM, CCC, lyophilized andshredded muscle tissue, collagen fibers, growth factors, antibiotics,stem cells, or other additives.

In making a strip, the dimensions are typically from about 10 mm to 500mm long, by 10 mm to 200 mm wide and 1 mm to 10 mm thick, more typicallyfrom about 15 mm to 200 mm long by 15 mm to 100 mm wide and 2 mm to 8 mmthick, even more typically from about 50 mm to 90 mm long by 15 mm to 35mm wide and 2 mm to 5 mm thick. The cross section of such a strip maytake any shape including a rectangle, square, triangle, other polygon,circle, half circle, ellipse, or partial ellipse.

For a sheet in this embodiment, the dimensions are typically from about20 mm to 300 mm by 10 mm to 100 mm and 1 mm to 10 mm thick, moretypically from about 30 mm to 150 mm by 20 mm to 70 mm and 1.5 mm to 8mm thick, even more typically from about 50 mm to 100 mm by 25 mm to 50mm and 2 mm to 5 mm thick.

For a film in this embodiment, the dimensions are typically from about20 mm to 300 mm by 10 mm to 100 mm and 0.1 mm to 5 mm thick, moretypically from about 30 mm to 150 mm by 20 mm to 70 mm and 0.25 mm to 3mm thick, even more typically from about 50 mm to 100 mm by 25 mm to 50mm and 0.5 mm to 1 mm thick. A film is characterized by removing mostair bubbles prior to or while molding a thin layer of material. The filmshape may be flat or may follow a 3D contoured shape. The film may becreated in a single layer or by laminating multiple layers of material.

For a disk in this embodiment, the dimensions are typically from about10 mm to 100 mm diameter and 1 mm to 10 mm thick, more typically fromabout 30 mm to 80 mm diameter and 2 mm to 8 mm thick, even moretypically from about 55 mm to 65 mm diameter and 2.5 mm to 5 mm thick. Adisk can be described as a cylinder with nominal diameter greater thannominal height.

For a molded 3D shaped object in this embodiment, the nominal outer bodydimensions are typically up to about 100 mm by 100 mm and 25 mm thick,more typically up to about 50 mm by 70 mm and 20 mm thick, even moretypically up to about 30 mm by 50 mm and 15 mm thick. A molded 3D shapeis typically defined so as to fit into a particular anatomical featureor surgically created space, such as a dental cavity, a drilled tunnelin a bone, or the space between two vertebral bodies in the spine.

For a plug in this embodiment, the dimensions are typically from about10 mm to 100 mm diameter and 1 mm to 10 mm tall, more typically fromabout 2 mm to 20 mm diameter and 5 mm to 50 mm tall, even more typicallyfrom about 4 mm to 15 mm diameter and 7 mm to 40 mm tall. A plug may bedescribed as a cylinder or extruded 2D shape, such as a square,triangle, star, or polygon, with nominal height greater than nominaldiameter or characteristic width of the 2D shape.

For a sponge in this embodiment, the dimensions are typically up toabout 500 mm by 500 mm and 50 mm thick, more typically up to about 100mm by 100 mm and 20 mm thick, even more typically up to about 50 mm by50 mm and 10 mm thick.

For a gasket in this embodiment, the outside body dimensions aretypically up to about 100 mm by 100 mm and 15 mm thick, more typicallyup to about 50 mm by 50 mm and 5 mm thick, even more typically up toabout 25 mm by 25 mm and 2.5 mm thick. The cross section of such agasket may take any shape including a rectangle, square, triangle, otherpolygon, circle, half circle, ellipse, or partial ellipse, tracing theentire periphery or some portion of the outside body shape.

It was unexpectedly discovered that the muscle-based implantablecomposition and tissue graft/implant of the present invention havehemostatic action and cause platelet activation. This is a particularadvantage during surgical procedures wherein patient bleeding is alwaysa problem. Thus, the muscle-based implantable composition and tissuegraft/implant of the present invention reduce the need for cauterizationat the interface between the patient's tissue and the implant.

The muscle-based composition and the tissue graft/implant of the presentinvention comprise a matrix that is formed from the fibers of defatted,shredded, allogeneic human muscle tissue. Due to the fibrous nature ofthe muscle tissues, the matrix that is formed is porous and particularlywell suited both for the infiltration by colonizing cells, and for thestorage and slow release of seeded cells, tissue growth factors, andchemotactic agents that attract a desired cell.

Thus, it is also within the scope of the present invention that thecomposition or implant/tissue graft of the present invention be combinedwith a natural or recombinant growth factor to stimulate tissue growthand healing, or a chemotactic agent to attract a desired cell. It isalso within the scope of the present invention that the composition ortissue graft/implant of the present invention be seeded with one or morecells prior to implantation into the patient. When the composition ortissue graft/implant of the present invention is to be used as a tissuegraft for skin, it is typically seeded with fibroblasts, preferably withmelanocytes. When the composition or tissue graft/implant of the presentinvention is to be used to treat a defect or an injury to a bone, it isseeded with osteogenic cells. Preferably, when the composition or tissuegraft/implant of the present invention is to be used to treat any tissuedefect or injury, it is seeded with stem cells that will provide thenative cells necessary for restoration of the injury or defect.

The tissue grafts and implants of the present invention exhibit a greatdegree of tensile strength. They are readily stitchable and retain amajority of their tensile strength even when rehydrated. In addition,upon hydration, tissue grafts and implants of the present invention aremoldable and suitable for filling in irregular gaps or holes in thetissue to be repaired. Typically, the hydrated tissue is press fitted bythe surgeon into the defect or cavity to be filled.

The implantable compositions and tissue grafts/implants of the presentinvention are formed from an intermediate composition comprising amuscle tissue slurry that is poured into a mold for formation into of acomposition or tissue graft/implant of any size or shape. As notedabove, the muscle tissue slurry can be combined with other agents, suchas DBM, CCC or a collagen (e.g., tendon, fascia) slurry before beingpoured into the mold. To produce an implantable film, a thin layer ofthe slurry is poured in a flat plate and the slurry is either air dried,air dried with positive airflow, or dried in an oven, preferably aconvection oven. To produce a sponge, a gasket or an implantable shape,the slurry (neat or amended) is poured into a mold of the appropriateshape, frozen (to retain its size), and lyophilized. The resulting driedimplantable shape is then ready for packaging and final sterilization.

In its second aspect, the present invention is directed to a method formaking a (muscle-based) composition or a tissue implant suitable fortreating an injury or a surgical or medical condition in a humanpatient, wherein the tissue implant comprises a matrix of digestedallogeneic human muscle. In this embodiment, the method comprises thesteps of:

-   -   i. removing the fat and soluble proteins from allogeneic or        xenogeneic mammalian muscle tissue;    -   ii. lyophilizing the muscle tissue from step (i);    -   iii. shredding the lyophilized muscle tissue;    -   iv. mixing the shredded muscle tissue in an aqueous carrier to        form a muscle tissue slurry having a viscosity within the range        of 1 centistoke to 20,000 centistokes;    -   v. transferring the muscle tissue slurry to an appropriate        shaped mold; and    -   vi. drying the slurry in the mold to form the correspondingly        shaped tissue implant.

In the above method, the matrix of digested allogeneic human musclecomprises from about 1% to about 100% of the final weight of thecomposition or implant, more typically from 50% to about 99% of thefinal weight of the implant, even more typically from 75% to about 99%of the final weight of the implant.

The muscle tissue slurry that is used to make the composition andimplant of the present invention is the subject of copending parentapplication, U.S. Ser. No. 10/754,310, filed Jan. 9, 2004, andincorporated herein by reference in its entirety. The muscle tissueslurry has a viscosity within the range of 1 centistoke to 20,000centistokes when measured at 25° C. Typically, the muscle tissue slurryhas a viscosity within the range of 1 centistoke to 10,000 centistokeswhen measured at 25° C.; more typically, the muscle tissue slurry has aviscosity within the range of 1 centistoke to 5,000 centistokes whenmeasured at 25° C.

The muscle tissue slurry that is used to make the composition and/ortissue graft/implants of the present invention is also characterizablein terms other than viscosity. Specifically, the muscle tissue slurry ischaracterized instead by the ratio of the volume of aqueous carrier(milliliters) to dry weight of muscle (grams). The aqueous carrier isacidic, basic or neutral. Preferably, the aqueous carrier is an aqueousacidic solution (“an acid”). Typically, the ratio of acid (volume) todry weight of muscle (grams) is within the range of 100:1 to 10:1; moretypically within the range of 80:1 to 20:1; most typically within therange of 70:1 to 30:1. The choice of the ratio of acid to proteindetermines the viscosity of the slurry and the choice is based upon theultimate application of the slurry. The muscle tissue slurry of thepresent invention was used to make the various tissue implants andgrafts (collectively “implants”) disclosed further herein.

The viscosity of the muscle tissue slurry (i.e., intermediatecomposition) ranges between slightly greater than the viscosity of waterto almost solid.

Although autogeneic muscle can be used in the intermediate compositionof the present invention, the source of the muscle is typically donormuscle that is obtained from cadavers and thus, the muscle isallogeneic. While the present invention is discussed herein in terms ofan allogeneic human muscle source and being used for preparing a tissueimplant for humans, any non-human mammal may be used as the muscle donorand the resulting slurry used to prepare a xenogeneic implant for use ina human or in another species of mammal. Preferred xenogeneic musclesources are porcine and bovine. Pigs are currently being used togenerate minimally antigenic hearts suitable for implantation as livingheart transplants in humans. In the examples herein, the applicantsdisclose that they ectopically implanted into a rat a tissue matrix (inthe form of a sponge) that was derived from human donor muscle and thetissue matrix was resorbed over a period of time without inducing aninflammatory response. This establishes the functionality of the tissuematrix as a biocompatible resorbable tissue scaffold even when implanted(as a xenograft) in a different species. Thus, there is evidence thatthe intermediate composition of the present invention would produce anacceptable tissue matrix even when made from xenograft muscle tissue.

In a preferred embodiment of the present invention, the muscle tissue isdefatted prior to or after being shredded. Preferably, it is defattedprior to being shredded. In this embodiment, the donor muscle is cutinto chunks of sufficiently small size (e.g., 20 mm×20 mm) to allow thetissue to be easily defatted. Suitable methods for defatting tissue arewell known in the art. Typically, this involves treating the tissue witha fat dissolving substance such as 60% to 90% alcohol in water. See U.S.Pat. No. 5,846,484, entitled “Pressure flow system and method fortreating a fluid permeable workpiece such as a bone,” which issued toScarborough, et al. on Dec. 8, 1998. In the present case, the applicantsutilized the assignees' well known method for defatting tissue, whichalso has the added benefit of removing blood, cellular debris, andsoluble and antigenic proteins, by subjecting the muscle tissue toalternating cycles of pressure and vacuum in the sequential presence ofsolvents, such as isopropyl alcohol, hydrogen peroxide and a detergent.These methods are disclosed in full detail in assignee's U.S. Pat. No.6,613,278, entitled “Tissue Pooling Process,” which issued to Mills etal., on 09/02/03; U.S. Pat. No. 6,482,584, entitled “Cyclic implantperfusion cleaning and passivation process,” which issued to Mills, etal. on Nov. 19, 2002; and U.S. Pat. No. 6,652,818, entitled “ImplantSterilization Apparatus,” which issued to Mills et al., on Nov. 25,2003, all of which are incorporated herein by reference in theirentirety.

In a more preferred embodiment, the defatted allogeneic human muscletissue was dried, more preferably by lyophilization, to facilitatefurther processing, such as shredding. The lyophilization process neednot remove all of the water. While the water content of the choppedallogeneic muscle can vary, it is typically dried to about 3% moisturecontent by weight. It is especially preferred that the allogeneic humanmuscle tissue be defatted prior to lyophilization.

The defatted and dried muscle tissue was shredded to a coarse fiber.Shredding is accomplished by any commercial shredder. Suitable shreddersinclude coffee grinders, food processors, and the like.

The shredded muscle tissue was mixed vigorously with a biocompatibleacid to produce the muscle slurry that is the intermediate compositionof the present invention. On a small scale, mixing was accomplished witha hand-held food processor. Mixing takes from 15 seconds to over twominutes and is dependent upon the amount of shredded protein and thevolume of acid. Mixing should continue until the slurry has uniformconsistency. After mixing, the slurry is preferably degassed. Degassingwas accomplished by centrifugation, or possibly vacuum centrifugation.

The biocompatible acid is either a biocompatible organic acid or abiocompatible inorganic acid. Preferably, a suitable biocompatible acidis selected from the group consisting of acetic acid, citric acid,formic acid, hydrochloric acid, lactic acid, phosphoric acid, phosphorusacid and sulfuric acid. More preferably, the biocompatible acid is anorganic acid; most preferably the organic acid is acetic acid. Thefunction of the acid is to partially digest the muscle tissue.

The intermediate composition employed in the present invention isimplantable in liquid form, such as by injecting into the patient at asite in need of restoration. In another embodiment, it is dried to apredetermined shape to prepare a variety of tissue implants.

Thus, the present invention is directed to a tissue graft of implantsuitable for treating an injury or a surgical or medical condition in ahuman patient, wherein the tissue implant ‘comprises a matrix ofdigested allogeneic human muscle. In this embodiment, the matrix ofdigested allogeneic human muscle comprises from about 1% to about 100%of the dry weight of the implant, more typically from 15% to about 95%of the dry weight of the implant, even more typically from 25% to about85% of the dry weight of the implant.

In another embodiment, the method of making a composition or tissueimplant of the present invention further comprises combining theallogeneic human muscle with a demineralized bone matrix (DBM),mineralized bone matrix, cortical cancellous chips (CCC), crushedcancellous chips, tricalcium phosphate, hydroxyapatite, or biphasiccalcium phosphate and/or shredded allogeneic human tendon. The additionof any of these components increases the viscosity of the intermediateslurry. When the tissue implant of the present invention contains DBM,mineralized bone matrix, CCC, crushed cancellous chips, tricalciumphosphate, hydroxyapatite, or biphasic calcium phosphate or acombination thereof, the resulting tissue implant is osteogenic andparticularly suited for repairing bone. The implant of this embodimentalso exhibits improved dimensional stability and ability to hold shapeduring drying, rehydration and handling. In one variation of the aboveembodiment, the DBM, mineralized bone matrix, CCC, crushed cancellouschips, tricalcium phosphate, hydroxyapatite, or biphasic calciumphosphate or a combination thereof are dispersed equally or randomlythroughout the matrix. In another variation, the DBM, mineralized bonematrix, CCC, crushed cancellous chips, or both are sandwiched betweenlayers of the matrix to form a laminate implant. In either of the abovereferenced embodiments, the DBM, mineralized bone matrix, CCC, crushedcancellous chips, tricalcium phosphate, hydroxyapatite, or biphasiccalcium phosphate or a combination thereof typically constitute from 1%to 99%, more typically from 50% to 99%, most typically from 90% to 99%of the dry weight of the composition or implant.

When the tissue implant contains tendon, it is tougher and stronger thanthe digested human muscle matrix alone and is particularly suited as adressing for a wound or burn that will become infiltrated with skincells and allow for development of a replacement skin layer that willcover the wound or burn. The ratio of tendon to intermediate compositionranges from 1:99 to 99:1 by dry weight. Typically, the range is 10:90 to90:10; more typically, the range is 25:75 to 75:25. While the abovediscussion is in relation to “tendon,” which is a preferred source ofcollagen for this invention, it is intended that any collagen source beused, including tendon, fascia and dermis.

After the mixing step, the resulting intermediate composition (thedigested allogeneic human muscle slurry) of the present invention isoptionally degassed, by pouring the slurry into plates or tubes, andcentrifuging them to eliminate any entrapped air and produce a higherdensity slurry.

Alternatively, the slurry is poured into a mold for formation of animplantable tissue matrix of any size or shape. As noted above, theslurry can be combined with other agents, such as DBM, CCC or a collagen(e.g., tendon, fascia) slurry before being poured into the mold. Toproduce an implantable film, a thin layer of the slurry is poured in aflat plate and the slurry is either air dried, air dried with positiveairflow, or dried in an oven, preferably a convection oven. To produce asponge, a gasket or an implantable shape, the slurry (neat or amended)is poured into a mold of the appropriate shape, frozen (to retain itssize), and lyophilized. The resulting dried implantable film or shape isthen ready for packaging and final sterilization.

Prior to implantation, the freeze dried composition, tissue graft orimplant of the present invention is removed from its sterile packagingand rehydrated by contacting them with water, saline, blood, plasma, abuffered solution, or any other suitable liquid. Preferably, therehydrating liquid contains a growth factor or a chemotactic agent asdiscussed above.

In a pilot study, prototype implants were resorbed into an ectopic sitein an athymic nude rat model, without any signs of an inflammatoryresponse. Specifically, an implant of Example 2, containing 20% DBM wasimplanted in abdominal muscle pouches of athymic nude rats using amodified Urist model. Urist, M. R., “Bone: Formation by Autoinduction,”Science 160:893-894 (1965). The explants were retrieved four weekslater, processed, and evaluated histologically for evidence of new boneformation. The control implants containing only the sponge carrier wereresorbed without evidence of inflammation. More significantly, theimplants containing DBM demonstrated signs of new bone formation(endochondral ossification). Hence, the muscle tissue matrix of thepresent invention, under the influence of DBM, provided scaffolding forcolonization by native restorative cells and the laying down of newbone.

Alternatively, the slurry is poured into a mold for formation of animplantable tissue matrix of any size or shape. As noted above, theslurry can be combined with other agents, such as DBM, mineralized bonematrix, CCC, crushed cancellous chips, or a collagen (e.g., tendon,fascia, or dermis) slurry before being poured into the mold. To producean implantable film, a thin layer of the slurry is poured in a flatplate and the slurry is either air dried, air dried with positiveairflow, or dried in an oven, preferably a convection oven. To produce asponge, a gasket or an implantable shape, the slurry (neat or amended)is poured into a mold of the appropriate shape, frozen (to retain itssize), and lyophilized. The resulting dried implantable film or shape isthen ready for packaging and final sterilization.

Several parameters are controlled during the molding process whichimpact the shape, composition, and material properties of the finishedgrafts. Shrinkage of the material during drying is noted, especially ascharacteristic thickness and bulk of the implant increase. Shrinkageresults in changes in shape, density, uniformity, thickness, appearance,and residual stresses of the implants. Shrinkage and its effects can becontrolled and manipulated by careful control of parameters includingdrying time, thickness of implant sections, shape of implant, coring orsegmenting of thick sections, and shape and configuration of molds.Other process parameters directly affecting the finished product includeorder of process steps, spatial orientation of the graft duringprocessing, and method of application of the slurry into the molds. Forexample, freezing before drying creates a thicker sponge implant withporous structure, where drying without freezing produces a compressedand much thinner film from a similar amount of graft material.Gravitational effects can produce grafts with material eitherconcentrated in a particular area or spread more evenly throughout themold. Thus, rotational molding can produce thin film grafts wrappedevenly around complex internal or external 3D molds, nominal dryingunder constant gravitational force can produce grafts which are slightlythicker at the bottom due to slow flowing of the slurry during drying,and drying in a vacuum centrifuge can produce grafts with materialstrongly concentrated in the direction of the centrifugal forces.Finally, different graft properties can be created by introducing theslurry to a given mold all at once in order to form a uniform implant,or by introducing it in layers or stages and allowing for drying oradding additional materials between layers to produce a layeredcomposite or fiberglass like structure.

Thus, in another embodiment, the present invention is directed to amethod for making the intermediate composition (muscle tissue slurry) ofthe present invention comprising the steps of:

-   -   i. removing the fat and soluble proteins from allogeneic or        xenogeneic mammalian muscle tissue;    -   ii. lyophilizing the muscle tissue from step (i);    -   iii. shredding the lyophilized muscle tissue; and    -   iv. mixing the shredded muscle tissue in an aqueous carrier to        form a muscle tissue slurry having a viscosity within the range        of 1 centistoke to 20,000 centistokes.

The tendon that is used in the tissue implants of the present inventionis processed the same as the allogeneic human muscle. It is chopped intopieces, defatted, freeze dried (lyophilized), shredded into a coarsefiber, and acid digested to provide a viscous tendon digestate that issuitable for combining with the acid digested allogeneic muscle(intermediate composition) of the present invention. The ratio of tendondigestate to intermediate composition ranges from 1:99 to 99:1.Typically, the range is 10:90 to 90:10; more typically, the range is25:75 to 75:25. While the above discussion is in relation to “tendon,”which is a preferred source of collagen for this invention, it isintended that any collagen source be used, including fascia. Thecollagen source is xenogeneic or allogeneic. Preferably, it isallogeneic.

Example 1 Preparation of a Slurry of Allogeneic Human Muscle

Skeletal muscle was removed from a donor cadaver and cut into chunks (20mm×20 mm). The chunks of skeletal muscle were defatted, deantigenizedand soluble protein was removed by subjecting the muscle tissue tocyclically alternating pressure and vacuum in the sequential presence ofthe isopropyl alcohol, hydrogen peroxide and a detergent. The method isfully described in assignee's U.S. Pat. No. 6,613,278, entitled “TissuePooling Process,” which issued to Mills et al., on Sep. 2, 2003; U.S.Pat. No. 6,482,584, entitled “Cyclic implant perfusion cleaning andpassivation process,” which issued to Mills, et al. on Nov. 19, 2002;and U.S. Pat. No. 6,652,818, entitled “Implant Sterilization Apparatus,”which issued to Mills et al., on Nov. 25, 2003, all of which areincorporated herein by reference in their entirety. After the abovecleansing process, the defatted and non-antigenic muscle tissue waslyophilized to remove the moisture. The lyophilization procedure was astandard 17-hour program. The dried chunks were shredded and chopped ina grinder. The processing time varied from 5 seconds to 2 minutesdepending upon the amount of lyophilized muscle being processed, drynessand starting size. At this stage, the shredded muscle tissue looks likefluffed fibers. The shredded muscle tissue were weighed and thencombined with a predetermined amount of 10% or 20% aqueous acetic acidaccording to the table below:

TABLE 1 Ratios of Acetic Acid (ml) to weight of dry muscle (g) Muscle(g) Muscle (g) Muscle (g) Acid Acid:muscle Acid:muscle Acid:muscle 10%acetic acid 0.5 g 0.75 g 1 g 46:1 34:1 22:1 20% acetic acid 0.5 g 0.75 g1 g 46:1 34:1 22:1

The combined acid solution and muscle tissue were mixed with a highspeed mixer until a uniform gel (i.e., slurry) was formed. Mixing tookbetween 15 seconds to more than 2 minutes depending upon the acidconcentration, amount of muscle tissue and volume of acid solution.

The above described slurry was used alone or combined with anothercomponent to make a tissue implant of the present invention. The lowerviscosity (more dilute) slurries were preferred when makingpourable/flowable films. The lower to intermediate viscosity slurrieswere more desirable when being combined with DBM or CCC or tendon, eachof which thickened the slurry.

Example 2 Formulation Comprising the Slurry of Example 1 and DBM

The slurry of Example 1 was degassed via centrifugation. After thedegassing, the slurry was transferred to a mixing bowl. DBM was addedand mixed to uniformity at ratios of 0.1%, 1%, 5%, 10%, 20% and 30% (DBMweight to slurry weight).

A portion of each of the above slurries containing the DBM were pouredinto molds and allowed to dry at room temperature with positive airflow.The dried products produced a series of muscle based tissue implants inthe form of films with the differing amounts of DBM embedded therein.

A second portion of the slurries from above was poured into molds,frozen, and lyophilized. The dried products produced a series ofsponge-like tissue implants having increasing amounts of DBM therein.

Example 3 Formulation Comprising the Slurry of Example 1 and CCC

The slurry of Example 1 was degassed via centrifugation. After thedegassing, the slurry was transferred to a mixing bowl. CCC was addedand mixed until uniform at a ratio of 50% (CCC volume to slurry volume).The slurry containing CCC was poured into a mold, frozen, andlyophilized. This dried product produced a tissue implant in the form ofa sponge with CCC imbedded therein.

The degassed slurry from above was transferred to a mixing bowl, where60% CCC (CCC volume to slurry volume) and 10% DBM (DBM volume to slurryvolume) were added to the degassed slurry with mixing. Mixing continueduntil a uniform appearing mixture was formed. The slurry was poured intoa cube shaped mold, frozen, and lyophilized. This dried product produceda cube shaped tissue implant having CCC and DBM imbedded therein.

Example 4 Formulation Comprising the Slurry of Example 1 and a Slurry ofTendon

The slurry from Example 1 was degassed via centrifugation. Afterdegassing, the slurry was transferred to a series of three (3) mixingbowls. Using allogeneic human tendon, a tendon slurry was made in theexact same manner as the muscle slurry. After the solubilized tendonslurry was degassed, a portion of it was added to each of the three (3)mixing bowls. The ratio of muscle to tendon in each of the three (3)bowls was 25:75, 50:50 and 75:25 (muscle volume:tendon volume),respectively. A portion of the tendon/skeletal slurry mixtures werepoured into flat molds and allowed to dry at room temperature withpositive airflow. Once dried, the three dried materials each produced atissue implant in the form of a film.

Three identical tendon/skeletal muscle slurries were poured into molds,frozen, and lyophilized. The lyophilized frozen slurries producedtendon/skeletal muscle based implants in the form of a sponge.

Example 5 Preparation of an Implantable Strip

A series of implantable strips were created from the films produced inExamples 1, 2, 3, and 4. Specifically, the dried films were cut intoimplantable strips that were 0.5 mm thick, 20 mm wide, and 70 mm long.

Example 6 Preparation of an Implantable Sheet

An implantable sheet was created from the films produced in Example 1,2, 3, and 4. The dried films either were left in the final shape oftheir molds, or were cut into sheets that were 0.5 mm thick, 70 mm wideand 70 mm long. Sheets were made that were also three dimensional, suchas convex and concave spherical bodies. These implantable threedimensional films were made via rotational molding, vacuumcentrifugation drying, room temperature drying, or room temperaturedrying with forced air, to a thin film in a three dimensional mold.Thicker sheets were produced in both two dimensional and threedimensional forms by successive reapplication of muscle tissue slurry,after drying of the preceding layer. In some cases DBM was added to theimplant between this application of successive layers, to create alaminated tissue implant impregnated with DBM.

Example 7 Preparation of an Implantable Sponge

A series of implantable sponges were made as specified in Examples 1, 2,3, and 4 above. Using appropriate molds, the sponges were made in asquare, circular, or hexagonal forms. The average thickness was 5 mm.The squares were as large as 50 mm by 50 mm. The circles had diametersas large as 90 mm. The hexagons were 40 mm per side.

Example 8 Preparation of an Implantable Gasket

Using the slurry of Example 1, a gasket was made as a thinner version(2.5 mm) of the sponge in Example 7. The gasket was used in conjunctionwith a bone plate to tie together two model vertebral bodies in thelaboratory.

Example 9 Preparation of a Graftable Wound Dressing

The films from Example 4 were suitable for use as a wound dressing/skingraft. The films are hydrated before use with sterile saline until softand pliable and then applied to the wound. Any excess film overhangingthe wound is cut off with surgical scissors.

Example 10 Biological Activity of an Ectopically Implanted Sponge in aRat

The implant of Example 2, containing 20% DBM, and measuring about 5 mmdiameter by about 25 mm length, was implanted in abdominal musclepouches of athymic nude rats using a modified Urist model. Urist, M. R.,“Bone: Formation by Autoinduction,” Science 160:893-894 (1965). Explantswere retrieved four weeks later, processed, and evaluated histologicallyfor evidence of new bone formation. Whereas control implants containingonly the sponge carrier were resorbed without evidence of inflammation,those containing DBM demonstrated signs of new bone formation(endochondral ossification).

Example 11 Composite Implants for Hip Replacement Applications

An implant following the method of Example 2, containing 20% DBM, waspoured into molds shaped to match an existing metal hip replacement stemgraft and an acetabular cup. The hip stem graft was structurallyenhanced with pieces of bone and bone fragments placed into the materialduring molding. The acetabular cup graft featured a two partconstruction, wherein a sponge implant section was formed inside of andbonded to an earlier formed, shaped three dimensional thin film mold. Noattempt was made to quantify the failure load or strength of theimplants, but the proof of concept objective was satisfied as the stemand cup implants both withstood normal handling and manipulation.

Example 12 Meniscus Replacement Implant

An implant following the method of Example 2, containing no DBM or DBMadded only to specific regions of the graft, is proposed as meniscusreplacement graft. The combination of precisely controlled shaping andcontouring with appropriate load bearing capacity and appropriateremodeling response with or without the application of specific growthfactors to various parts of the graft answers a unique need in the art.

Example 13 Precise Anatomically Matched Implants

An implant following the method of Example 2, can be formed in a moldcreated by known methods from digitized data of an area in need oftreatment. Methods of mold creation are known in the art, and caninclude x-ray, ultrasound, or cat-scan imaging data translated to acomputer aided design (CAD) or computer aided manufacturing (CAM) orcomputer numerical controlled (CNC) machining center. The implants madefrom these methods could be used to treat conditions where exactmatching of the anatomical structure is critical and where abiocompatible allograft implant is beneficial. Applications can includefacial reconstruction, general orthopedic defects and bone segmentreplacements, and vertebral body replacements.

Example 14 Flowable Implants

An implant following the method of Example 2 was transferred to asyringe after drying and full rehydration in sterile saline solution.The rehydrated implant was found to be flowable under the forces of thesyringe, and could be placed in rows or used to fill cavities similar tothose found commonly in surgical sites. A second flowable implant wascreated by transferring the slurry of Example 1 directly into a syringeand then using the syringe to flow the graft material into rows or intocavities similar to those found commonly in surgical sites.

Example 15 Moldable Implants

An implant following the method of Example 2 was partially rehydratedwith sterile saline solution after drying. The implant was placed into acavity and molded to the shape of the cavity under manual manipulation.The implant molded to the shape of the cavity and then retained thatshape under minimal forces including gravity and light rinsing.

1-18. (canceled)
 19. An intermediate composition for producing animplant suitable for implantation in humans comprising a matrix ofshredded allogeneic or xenogenic tissue that has been combined with anaqueous carrier to form a tissue slurry having a viscosity within therange of 1 centistoke to 20,000 centistokes at 25° C.
 20. Theintermediate composition of claim 19, wherein said tissue slurry has aviscosity within the range of 1 centistoke to 10,000 centistokes at 25°C.
 21. The intermediate composition of claim 20, wherein said tissueslurry has a viscosity within the range of 1 centistoke to 5,000centistokes at 25° C.
 22. The intermediate composition of claim 19,wherein said shredded allogeneic tissue is also defatted.
 23. Theintermediate composition of claim 19, wherein the ratio of aqueouscarrier in milliliters to tissue in grams is within the range from 80:1to 20:1.
 24. The intermediate composition of claim 23, wherein the ratioof aqueous carrier in milliliters to tissue in grams is within the rangefrom 70:1 to 30:1.
 25. The intermediate composition of claim 19, whereinsaid shredded allogenic tissue is muscle, tendon, fascia, ligament ordermis.
 26. The intermediate composition of claim 19, wherein thecomposition further comprises particles of demineralized bone matrix(DBM), mineralized bone matrix, cortical cancellous chips CCC, crushedcancellous chips, tricalcium phosphate, hydroxyapatite, or biphasiccalcium phosphate dispersed in the matrix.
 27. The intermediatecomposition of claim 26, wherein the composition comprises demineralizedbone matrix (DBM).
 28. The intermediate composition of claim 19, furthercomprising a bone morphogenic protein (BMP), a LIM mineralizationprotein, a RUNX-2 protein, a transforming growth factor-α, atransforming growth factor-β, a fibroblast growth factor, a plateletderived growth factor, an epidermal growth factor, an insulin-likegrowth factor, or a combination thereof.
 29. The intermediatecomposition of claim 28, wherein said BMP is selected from the groupconsisting of BMP-2, BMP-4 and BMP-7.
 30. The intermediate compositionof claim 19, wherein the composition further comprises seeded cells, agrowth factor, a chemotactic agent, or a combination thereof.
 31. Theintermediate composition of claim 19, wherein said composition comprisesa growth factor suitable for inducing the growth of fibroblasts,melanocytes, osteogenic cells, stem cells, skin cells or a combinationthereof.
 32. The intermediate composition of claim 30, wherein saidcomposition comprises seeded cells that are dermatocytes, fibroblasts,melanocytes, osteogenic cells, stem cells, skin cells or a combinationthereof.
 33. The intermediate composition of claim 19, which has beentreated to remove or neutralize antigenic proteins, bacteria andviruses.
 34. The intermediate composition of claim 19, wherein saidaqueous carrier comprises a biocompatible acid selected from the groupconsisting of acetic acid, citric acid, formic acid, hydrochloric acid,phosphoric acid, phosphorus acid and sulfuric acid.
 35. An implantsuitable for treating an injury or a medical condition in a humancomprising the intermediate composition of claim
 19. 36. The implant ofclaim 35, wherein said implant is implantable in liquid form.
 37. Theimplant of claim 35, wherein said implant forms a scaffold material fortissue regeneration in humans.
 38. A method for making a tissue slurrycomprising the steps of: i. removing the fat and soluble proteins fromallogeneic or xenogenic tissue; ii. lyophilizing the muscle tissue fromstep (i); iii. shredding the lyophilized muscle tissue; and iv. mixingthe shredded muscle tissue in an aqueous carrier to form a tissue slurryhaving a viscosity within the range of 1 centistoke to 20,000centistoke.